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Ocean Science Journal

, Volume 53, Issue 1, pp 81–90 | Cite as

Genus-specific PCR Primers Targeting Intracellular Parasite Euduboscquella (Dinoflagellata: Syndinea)

  • Jae-Ho Jung
  • Jung Min Choi
  • Young-Ok Kim
Article

Abstract

We designed a genus-specific primer pair targeting the intracellular parasite Euduboscquella. To increase target specificity and inhibit untargeted PCR, two nucleotides were added at the 3’ end of the reverse primer, one being a complementary nucleotide to the Euduboscquella-specific SNP (single-nucleotide polymorphism) and the other a deliberately mismatched nucleotide. Target specificity of the primer set was verified experimentally using PCR of two Euduboscquella species (positive controls) and 15 related species (negative controls composed of ciliates, diatoms and dinoflagellates), and analytical comparison with SILVA SSU rRNA gene database (release 119) in silico. In addition, we applied the Euduboscquella-specific primer set to four environmental samples previously determined by cytological staining to be either positive or negative for Euduboscquella. As expected, only positive controls and environmental samples known to contain Euduboscquella were successfully amplified by the primer set. An inferred SSU rRNA gene phylogeny placed environmental samples containing aloricate ciliates infected by Euduboscquella in a cluster discrete from Euduboscquella groups a–d previously reported from loricate, tintinnid ciliates.

Keywords

marine parasite syndinean dinoflagellate SNP target-specific PCR primer ciliates 

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References

  1. Akselman R, Santinelli N (1989) Observaciones sobre dinoflagelados parasitos en el litoral Atlantico sudoccidental. Physis B Aires Sec A 47:43–44Google Scholar
  2. Bachvaroff TR, Kim S, Guillou L, Delwiche CF, Coats DW (2012) Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis. Appl Environ Microb 78:334–345CrossRefGoogle Scholar
  3. Bui M, Liu Z (2009) Simple allele-discriminating PCR for costeffective and rapid genotyping and mapping. Plant Methods 5:1CrossRefGoogle Scholar
  4. Cachon J (1964) Contribution à l’étude des péridiniens parasites. Cytologie, cycles évolutifs. Ann Sci Nat Zool 6:1–158Google Scholar
  5. Campbell AS (1927) Studies on the marine ciliate Favella (Jörgensen), with special regard to the neuromotor appartus and its rôle in the formation of the lorica. Univ Calif Publ Zool 29:429–452Google Scholar
  6. Chatton É (1952) Classe des dinoflagellés ou péridiniens. In: Grassé P-P (ed) Traité de zoologie. Masson & Cie, Paris, pp 309–390Google Scholar
  7. Coats DW, Bachvaroff TR (2013) Parasites of tintinnids. In: Dolan JR, Montagnes DJS, Agatha S, Coats DW, Stoecker DK (eds) The biology and ecology of tintinnid ciliates: models for marine plankton. John Wiley, Chichester, pp 145–170Google Scholar
  8. Coats DW, Kim YO, Choi JM, Lee ES (2014) Observations on dinoflagellate parasites of aloricate ciliates in Korean coastal waters. Aquat Microb Ecol 72:89–97CrossRefGoogle Scholar
  9. Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: more models, new heuristics and parallel computing. Nat Methods 9:772CrossRefGoogle Scholar
  10. Dunthorn M, Klier J, Bunge J, Stoeck T (2012) Comparing the hyper-variable V4 and V9 regions of the small subunit rDNA for assessment of ciliate environmental diversity. J Eukaryot Microbiol 59:185–187CrossRefGoogle Scholar
  11. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26:2460–2461CrossRefGoogle Scholar
  12. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol 59:307–321CrossRefGoogle Scholar
  13. Hall T (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acid S 41:95–98Google Scholar
  14. Harada A, Ohtsuka S, Horiguchi T (2007) Species of the parasitic genus Duboscquella are members of the enigmatic marine alveolate group I. Protist 158:337–347CrossRefGoogle Scholar
  15. Jung J-H, Choi JM, Coats DW, Kim Y-O (2016) Euduboscquella costata n. sp. (Dinoflagellata, Syndinea), an intracellular parasite of the ciliate Schmidingerella arcuata: morphology, molecular phylogeny, life cycle, prevalence, and infection intensity. J Eukaryot Microbiol 63:3–15CrossRefGoogle Scholar
  16. Jung J-H, Kim S, Ryu S, Kim M-S, Baek Y-S, Kim S-J, Choi J-K, Park J-K, Min G-S (2012) Development of single-nucleotide polymorphism (SNP)-based phylum-specific PCR amplification technique: Application to the community analysis using ciliates as a reference organism. Mol Cells 34:383–391CrossRefGoogle Scholar
  17. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Mentjies P, Drummond A (2012) Geneious Basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics 28:1647–1649CrossRefGoogle Scholar
  18. Kilias ES, Nöthig E-M, Wolf C, Metfies K (2014) Picoeukaryote plankton composition off West Spitsbergen at the entrance to the Arctic Ocean. J Eukaryot Microbiol 61:569–579CrossRefGoogle Scholar
  19. Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16:111–120CrossRefGoogle Scholar
  20. Lee ES, Xu D, Shin MK, Kim Y-O (2012) First record of six marine ciliate species of genus Strombidium (Ciliophora: Spirotricha: Oligotrichia) from Korea with ecological notes. Anim Syst Evol Diversity 28:192–207CrossRefGoogle Scholar
  21. Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491–499CrossRefGoogle Scholar
  22. Montagnes DJS, Lynn DH (1987) A quantitative protargol stain (QPS) for ciliates: method description and test of its quantitative nature. Mar Microb Food Webs 2:83–93Google Scholar
  23. Quast C, Pruesse E, Yilmaz P, Gerken J, Schweer T, Yarza P, Peplies J, Glöckner FO (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596CrossRefGoogle Scholar
  24. Ronquist F, Huelsenbeck JP (2003) MrBayes 3: bayesian phylogenetic inference under mixed models. Bioinformatics 19:1572–1574CrossRefGoogle Scholar
  25. Sebastián CR, O’Ryan C (2001) Single-cell sequencing of dinoflagellate (Dinophyceae) nuclear ribosomal genes. Mol Ecol Notes 1:329–331CrossRefGoogle Scholar
  26. Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739CrossRefGoogle Scholar
  27. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680CrossRefGoogle Scholar

Copyright information

© Korea Institute of Ocean Science & Technology (KIOST) and the Korean Society of Oceanography (KSO) and Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  1. 1.South Sea Environment Research Center, South Sea Research InstituteKIOSTGeojeKorea
  2. 2.Department of Biology, College of Natural SciencesGangneung-Wonju National UniversityGangneungKorea

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